EP2918284B1 - Polypeptides dérivés de tgf et leurs utilisations - Google Patents

Polypeptides dérivés de tgf et leurs utilisations Download PDF

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EP2918284B1
EP2918284B1 EP13805228.7A EP13805228A EP2918284B1 EP 2918284 B1 EP2918284 B1 EP 2918284B1 EP 13805228 A EP13805228 A EP 13805228A EP 2918284 B1 EP2918284 B1 EP 2918284B1
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polypeptide
tgfβ
prt
artificial sequence
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EP2918284A1 (fr
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Ángel de Jesús CORRIA OSORIO
Kalet LEÓN MONZÓN
Tania Carmenate Portilla
Amaury PUPO MERIÑO
Saumel PÉREZ RODRÍGUEZ
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Centro de Immunologia Molecular
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/495—Transforming growth factor [TGF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1841—Transforming growth factor [TGF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/04—Drugs for skeletal disorders for non-specific disorders of the connective tissue
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the present invention relates to the fields of Biotechnology and Medicine. Particularly to the use of mutated variants of TGF ⁇ molecule, which are antagonists of the signaling of their natural counterparts, and to the therapeutic application of these variants.
  • TGF ⁇ cytokines were discovered by their capacity to stimulate cell colony formation ( Roberts AB, et al, Proc Natl Acad Sci USA 78: 5339-43, 1981 ); because this process is a classic marker of cellular transformation, this molecule was called Transforming Growth Factor beta.
  • TGF ⁇ ligands TGF ⁇ 1, TGF ⁇ 2, TGF ⁇ 3 are recognized as the prototype of multifunctional growth factors. Almost any type of cell in the body produces them and expresses its receptor complex. These molecules are potent inhibitors of the proliferation of epithelial, endothelial and hematopoietic cells ( Miyazono, K., J.
  • TGF ⁇ signaling is a very important regulation node.
  • One of its most important functions is maintaining lymphocyte homeostasis and immune tolerance, through inhibition of the proliferation of na ⁇ ve T cells induced by self-antigens in lymphopenic environments.
  • This molecule also suppresses or alters the activation, maturation and differentiation of natural killer cells ( Laouar, Y. et al Nature Immunol 6: 600-607, 2005 ), dendritic cells ( Luo, X. et al, Proc. Natl Acad .
  • the TGF ⁇ plays an essential role in the induction, differentiation and maintenance of natural and induced regulatory T cells (CD4 +Foxp3+) and TCD4+IL17+ (TH17) effector T cells ( Kryczek, I. et al, J. Immunol. 178 : 6730-33, 2007 ; Moo-Young, TA et al. J. Immunother 32: 12-21, 2009 ; Fantini, MC et al J. Immunol. 172: 5149-53, 2004 ; Flavell, R.A. Cell 134: 392-404, 2008 ).
  • Mature TGF ⁇ ligands are homodimers of 112 amino acid residues. They are derived from a precursor molecule formed by the latency associated pro-peptide (LAP) located at N-terminal and the active domain towards the C-terminus extreme. Both domains are intracellularly separated by proteolysis and the ligands are secreted as inactive precursors, formed by the prodomain reversibly bound to the active domain, thus regulating access to cellular receptors ( Geoffrey D. Young and Joanne E. Murphy-Ullrich. JBC Vol 279, No. 36: 38032-39, 2004 ). It has been postulated that the pro-peptide associated to latency is also important for secretion of the mature domain ( Gray A. and A Mason Science 247:1328-30, 1990 ).
  • TGF ⁇ 1, TGF ⁇ 2, TGF b3 interact in the plasma membrane with receptors T ⁇ RI, T ⁇ RII and T ⁇ RIII.
  • the latter also known as Betaglycan, is not expressed in all cell types, and although it is dispensable for the signaling mediated by TGF ⁇ 1 and TGF ⁇ 3 ligands, constitutes a reservoir of these ligands when T ⁇ RII is saturated.
  • T ⁇ RIII forms complexes with T ⁇ RI and T ⁇ RII receptors presenting the ligand to them.
  • TGF ⁇ 1 and TGF ⁇ 3 can bind to T ⁇ RII with high affinity (5-30pm), while TGF- ⁇ 2 can only do it in the same way in the presence of T ⁇ RIII ( De Crescenzo et al, J Biol Chem 279: 26013-18, 2004 ; De Crescenzo et al, J. Mol. Biol 328: 1173-1183, 2003 ; Groppe et al, Molecular Cell 29, 157-168, 2008 ).
  • T ⁇ RII belongs to ( Huang F and YG Chen Cell Biosc Mar 15, 2 : 9, 2012 ). However they can bind with several type I receptors.
  • ALK5 is described as the reference T ⁇ RI receptor of its ligand subfamily. After its recruitment into the T ⁇ RII/TGF- ⁇ complex the phosphorylation of SMAD2/3 proteins is induced ( Huang F and YG Chen Cell Biosc; Mar 15; 2:9, 2012 ).
  • ALK1 is activated in response to the formation of the TGF/ T ⁇ RII complex in endothelial cells and signals by SMAD1 and SMAD5 ( Goumans MJ et al, Mol Cell 12 (4): 817-28, 2003 ). In some epithelial cells the SMAD1/5 signaling is induced by receptor ALK2, ALK3 and ALK6 ( Daly AC et al, Mol Cell Biol, 28: 6889-6902, 2008 ). ALK2 is also associated with processes related to in vivo cardiovascular development ( Olivey HE et al, Dev Dyn 235 (1): 50-9, 2006 ).
  • T ⁇ RII and ALK5 are unique to TGF ⁇ 1, TGF ⁇ 2 and TGF ⁇ 3 family of ligands while ALK1/2/6/3 are more promiscuous and also shared by Activins and Bone Morphogenetic Proteins ( Sebald W. et al, Biol Chem, 385 (8): 697-710, 2004 ).
  • ALK5 mediated signaling is associated with various pathogenic mechanisms in certain diseases.
  • cancer for example, its role is complex and is associated with the suppression of immune response and promotion of tumor progression.
  • the suppression of immune response occurs mainly during early stages of tumorigenesis. While its role as tumor progressor occurs in advanced stages of the carcinogenesis, through the induction of metastatic invasive phenotypes and the suppression of anti-tumor immune response ( Miyazono K. Nat Rev. Cancer 10: 415-24, 2010 ; Miyazono K. Cancer Sci 101 (2): 306-12, 2010 ; Hawinkels LJ. et al, Growth Factors 29(4):140-52, 2011 ).
  • TGF ⁇ Another illness in which of TGF ⁇ 's activity is also deleterious are chronic infections caused by pathogens such as HIV, HBV, HCV, CMV, mycobacteria and Trypanosoma cruzi parasite.
  • pathogens such as HIV, HBV, HCV, CMV, mycobacteria and Trypanosoma cruzi parasite.
  • the TGF ⁇ exerts a negative influence on the protective immune response, allowing growth and survival of these intracellular pathogens ( Reed GS. Microbes and Infection 1: 1313-1325, 1999 ).
  • In many diseases overproduction of TGF ⁇ contributes to pathological excess of fibrotic tissue ( Santiago B. et al., J Invest Dermatol 125: 450-455, 2005 ), which compromises the normal function of the damaged organ.
  • fibrotic tissues Some examples of pathological excess of fibrotic tissues are pulmonary fibrosis, sarcoidosis, cardiac fibrosis, cardiomyopathy, liver cirrhosis, systemic sclerosis, glomerular sclerosis and primary biliary cirrhosis, among others ( Kopp JB et al, Microbes and Infection, 1: 1349 - 1365, 1999 ).
  • the present invention is related to the scientific finding that mutated variants of the TGF ⁇ family (TGF ⁇ 1, TGF ⁇ 2 and TGF ⁇ 3) may inhibit the function of their natural counterparts.
  • the inventors found for the first time in in vitro experiments that the mutated variants of TGF ⁇ can substantially inhibit the signaling induced by native variants and thus neutralize its biological effects. This finding provides the basis of a new strategy for modulating TGF ⁇ signaling in diseases such as cancer, fibrosis or chronic infection in which these ligands have deleterious activity.
  • the present invention is about polypeptides with high homology at the level of primary sequence with the human TGF ⁇ , except in some positions where the native amino acid have been mutated to eliminate or to decrease substantially their ability to signal throught the ALK5 type 1 receptor.
  • These mutated variants of TGF ⁇ maintain its capacity to bind with high affinity to T ⁇ RII and T ⁇ RIII receptors, but are unable to signal because they do not interact with the ALK5 type I receptor, negatively modulating natural variants signaling by competing with them for binding to high affinity T ⁇ RII and T ⁇ RIII receptors.
  • the invention also includes the therapeutic applications of these mutated variants, alone or in combination with vaccines or other therapeutic modalities for the treatment of diseases such as cancer, fibrosis or chronic infections in which the action of TGF ⁇ influences the course of the disease.
  • the novelty of the present invention is that it provides a novel therapeutic approach to modulate TGF ⁇ signaling and thus modulate the invasive and metastatic capacity of various tumor and the activity of various immune system and connective tissue cells, in diseases where the function of TGF ⁇ reduce the protective immune response either natural or induced by vaccination or inducing an excessive tissue repair and fibrosis. None of the inhibition strategies mentioned above uses muteins of TGF ⁇ ligands as signaling antagonists. TGF ⁇ mutants of the present invention are virtually self-proteins and therefore have low immunogenicity, which minimizes the risk of antibody responses against them. Their high specificity for the TGF ⁇ receptor system reduces unexpected toxicities (common in strategies based in small size inhibitors).
  • TGF ⁇ mutated variants of TGF ⁇ maintain binding affinities to T ⁇ RII receptor at least as native TGF ⁇ 1 and TGF ⁇ 3 (6-10 pM). This affinity is hard to achieve with other receptor or ligand inhibition strategies, with monoclonal antibodies or other drugs. Surprisingly the authors of the present invention found that these TGF ⁇ mutated variants maintain their ability to interact with T ⁇ RII and T ⁇ RIII receptor, which constitutes an advantage over T ⁇ RII anti-receptor antibodies, since muteins block all possible binding sites of natural ligands to cell surface.
  • the present invention relates to polypeptides that antagonize the activity of TGF ⁇ natural ligands mediated by the ALK5 receptor as described in claim 1. These polypeptides possess homology greater than 90% with respect to the amino acid sequence of TGF ⁇ natural ligands.
  • the polypeptides of the present invention have at least one mutation in their primary amino acid sequence, in one of the residues selected from the group consisting of Y6, W30, W32, 151, L51, Q67 and L101.
  • the original amino acid residue is mutated by one of amino acid residues selected from the group comprising; for position 6: A for position 30: N, R, K, D, Q, L, S, P, V, I, G, C, T, A, E; for position 32: A; for position 51: Q, W, Y, A; for position 67: H, F, Y, W and for position 101: A, E; It is another object of the present invention polypeptides in which mutations were added in the interaction interface with receptors TGF ⁇ RII and/or TGF ⁇ RIII increasing its affinity to them.
  • the present invention relates to pharmaceutical compositions used for the treatment of cancer, diseases accompanied by fibrosis and chronic infectious diseases that contain one or a mixture of the polypeptides described in the present invention and a pharmaceutically suitable vehicle for their administration.
  • Such polypeptides may be covalently linked to a carrier protein, which may be Albumin or the Fc region of human immunoglobulins.
  • the polypeptides may be pegylated.
  • the present invention relates to the use of the polypeptides described to manufacture a pharmaceutical composition, useful in the treatment of cancer, diseases accompanied by fibrosis and chronic infectious diseases, as well as to enhance the cellular and/or humoral response to vaccines in replacement of native TGF ⁇ especially when the vaccine to potentiate is a therapeutic vaccine for cancer treatment.
  • the present invention relates to polypeptides having 112 amino acids in length. These polypeptides maintain a high sequence identity with the different native TGF ⁇ molecules, more than 90% identity. In a zone of their sequence, the polypeptides include 1-6 mutations as compared to native TGF ⁇ . The residues that replace the original residues are selected to have very different physicochemical properties with respect to the original amino acid, a change in the residue from nonpolar to polar, from charged to uncharged, from large to small and from acid to basic.
  • TGF ⁇ muteins polypeptides, which may also be called TGF ⁇ muteins, were designed based on the three dimensional structure of native TGF ⁇ 1, TGF ⁇ 2 and TGF ⁇ 3 in complex with their receptors T ⁇ RII and ALK5 (and entered in the PDB database). Mutations were introduced only at TGF ⁇ positions corresponding to amino acids significantly exposed to the solvent that form part of the ALK5 receptor binding region but not that of T ⁇ RII receptor. These residues were predicted to be important in the interaction with ALK5 using bioinformatic programs of public domain. The following table shows residues from the binding interface to ALK5 that are predicted to be important in the interaction and the possible mutations that affect the binding.
  • polypeptides of this invention can be obtained by various procedures including chemical synthesis of proteins. They may also be obtained by genetic engineering techniques, such as expressing them as inclusion bodies in bacteria such as E. coli or in mammalian cells such as CHO and HEK293 using any transfection protocol reflected in the art of the technique. Mutations at specific positions may also be obtained by directed mutagenesis techniques directed by means of reaction of the polymerase chain.
  • polypeptides of the present invention are selected from the in vitro and in vivo experiments to simultaneously possess the following properties:
  • the present invention encompasses additional modifications of the TGF ⁇ muteins from which the mature domain lacking LAP could be produced, without affecting the secretion thereof. This entails that the muteins can be obtained in their active form so that they interact with T ⁇ RII, which results in the simplification of the production process.
  • the present invention relates to additional modifications of TGF ⁇ muteins that increase their half-life.
  • modifications include pegylation, the fusion of any of the above-described immunomodulatory polypeptides to a carrier protein, which can be Albumin or the Fc region of human immunoglobulins, among others.
  • the present invention relates to mutated variants of TGF ⁇ (specific mutations are referred to in Table 2) fused to an Fc fragment of a human IgG1 which results in a mutein that has been selected to display the properties referred to above.
  • the Fc region selected for the coupling has a set of mutations (ala234, ala235) on the C ⁇ 2 domain that prevent it from interacting with Fc gamma receptors, except with the neonatal Fc receptor, thus silencing its ability to induce immune effector mechanisms ( Labrijn AF et al, Curr Opin Immunol. Aug, 20 (4):479-85, 2008 . Epub 2008 Jul 9).
  • the present invention also encompasses additional modifications of TGF ⁇ muteins, made in order to either increase their affinity to T ⁇ RII and T ⁇ RIII, but without affecting or even improving their inhibitor character, or to improve their in vivo pharmacodynamic with the increase of half-life.
  • additional mutations may be obtained by rational design with bioinformatics tools or using combinatorial molecular libraries of different nature (phage display libraries, libraries of gene expression in yeast or bacteria).
  • This invention also includes the pharmaceutical compositions comprising as active principle TGF ⁇ muteins and their analogs or the fusion proteins disclosed by the present invention as well as their possible therapeutic applications for the modulation of the invasive and metastatic capacity of various tumors and the activity of various immune system and connective tissue cells in diseases in which the functions of TGF ⁇ reduce the protective immune response, either natural or induced by vaccination, or induce an excessive tissue repair and fibrosis.
  • the polypeptides of the present invention can be administered to a subject carrying a disease independently or combined with other polypeptides or other substances that facilitate or enhance its therapeutic action.
  • the administration route may be any of administration routes described in the art of the technique for parenteral administration of drugs.
  • the polypeptides may be preferably administered intravenously, intramuscularly, subcutaneously or intratumorally.
  • polypeptides or fusion proteins described in the present invention can also be administered as part of a pharmaceutical composition useful in the therapy of cancer, diseases accompanied by fibrosis and chronic infectious diseases.
  • the present invention encompasses compositions, including pharmaceutical compositions, comprising a pharmaceutically acceptable vehicle.
  • the composition includes pharmaceutically acceptable carriers.
  • the pharmaceutically acceptable carriers include, but are not limited to: saline, sterile water, phosphate buffered saline, and similars. Other buffering agents, dispersed agents and inert non-toxic substances suitable for administration to a patient may be included in the compositions of the present invention.
  • the compositions may be appropriate solutions for the administration and are sterile and free of unwanted particles.
  • the present invention also relates to methods of treatment comprising the administration of a therapeutically effective amount of polypeptides, fusion proteins or compositions described in this invention to subjects with cancer, diseases accompanied by fibrosis or chronic infectious diseases.
  • the subject is a human being.
  • polypeptides or fusion proteins described by the present invention may also be administered in combination with traditional oncological therapies (chemotherapy and radiation) as an enhancer of the effect of these.
  • the polypeptides of the present invention should be administered at doses high enough to guarantee that their concentration in the lymph node or peripheral site is appropriate for the disease of interest, that is, in the concentration range in which mutein shows an inhibitory effect of native TGF ⁇ .
  • the dose must therefore be adjusted to the type of disease and route of administration being studied.
  • the dose should be adjusted to achieve mutant concentrations inside the tumor and/or loco-regional lymph node high enough to exhibit an inhibitory effect.
  • Dose ranges to be explored may vary between 1.25-20 mg/kg/dose weekly or biweekly.
  • the number of administrations should also be adjusted to the biodistribution of the mutein in question.
  • the aforementioned effective concentrations should be sustained for a time period ranging from 2 to 30 consecutive days. If the mutein is coupled to a carrier protein, the frequency of administration should be adjusted accordingly.
  • the present invention provides, for example, concurrent, staggered or alternated treatment options.
  • the compound/s of the invention may be administered simultaneously in separate pharmaceutical compositions, that is, a compound of the invention may be administered before or after the other anticancer agent, for example, with a difference of seconds, minutes, hours, days or weeks.
  • Therapeutic action is defined as full or partial remission of the symptoms of a disease.
  • therapeutic action is defined as a decrease in tumor volume or increase in relapse time, amongst others.
  • the TGF ⁇ mutants were designed computationally using bioinformatics techniques. As starting point the reported structures of the ternary complex of TGF ⁇ 1 and TGF ⁇ 3 with the TGF ⁇ receptor were used. The energy of binding of the native isoforms and all possible mutated variants was determined using public bioinformatics programs.
  • G_M1 mutein were expressed in the CHO-K1 cells using a genetic construct based in the lentiviral vector PLW that included the C-terminal hinge region and the domains C ⁇ 2 and Cy3 of a human IgG1 and a histidine tail. G_M1 was purified by affinity chromatography with protein A. It was obtained ( Figure 1 ) with high purity (> 95%).
  • Example 2 Evaluation of the binding of the mutein G_M1 to T ⁇ RII by ELISA.
  • Example 3 Mutein G_M1 have a reduced capacity to signal through receptor T ⁇ RI (ALK5), and therefore to mimic the native TGF ⁇ biological activity.
  • Example 4 Mutein G_M1 is antagonist of TGF ⁇ signaling in vitro.
  • CTLL-2 cells/wells were stimulated with rIL-2 (50U/ml) and cultured in the presence of 2 pM of TGF ⁇ 1 wild type and indicated concentrations of G_M1, or isotype control hR3 MAb; or anti-TGF ⁇ 1 antibody as a positive control. After 48 hour, alamar blue reagent was added to the culture and its reduction was measured at 540 and 630nm.
  • the G_M1 mutein but not the isotype control MAb hR3, neutralizes the inhibition of IL2-dependent CTLL-2 cell line proliferation, caused by native TGF ⁇ 1 ( Figure 4 ).
  • the neutralizing effect caused by the mutein is similar to that obtained with an anti-TGF ⁇ 1 antibody, which was used as positive control of the experiment.
  • Example 6 The mutein G_M1 inhibits the differentiation of naive CD4+ T cells into regulatory Foxp3+ CD4+T cells induced by native TGF ⁇ 1 and IL2.
  • CD4+CD25-GITR- naive population from the spleen of 4 C57/BL6 mice was purified by cells sorting. 5x10 4 of these cells were stimulated with 3 ⁇ g/mL plate bound anti-CD3 and 3 ⁇ g/mL of soluble anti-CD28, in presence of 5 ng/ml IL-2 and 5 ng/ml of native human TGF ⁇ 1. In this culture condition the percentage of regulatory T cells recovered after 3 days was of 55.6%. The effect of the mutein in this culture was evaluated at concentrations of 250 and 500ng/mL. The percentage of Foxp3+ regulatory T cells recovered, in the presence or absence of the mutein was measured. Figure 6 shows how the percentage of regulatory T cells recovered in the T cell cultures is reduced almost to zero in the presence of G_M1 mutein.

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Claims (13)

  1. Polypeptide mutant de TGFβ qui antagonise l'activité des ligands naturels de TGFβ médiée par le récepteur ALK5, ledit polypeptide ayant plus de 90 % d'identité avec la séquence de
    (i) TGFβ1 humain natif,
    (ii) TGFβ2 humain natif, ou
    (iii) TGFβ3 humain natif ;
    et dans lequel ledit polypeptide comprend au moins une mutation de substitution dans l'un des résidus d'acides aminés choisis dans le groupe comprenant W30, W32, L101, I51, L51, Q67 et Y6 de la séquence de TGFβ1, TGFβ2 ou TGFβ3 humain natif, dont la numérotation des résidus d'acides aminés est fournie dans l'une quelconque des SEQ ID NO : 2 à 73.
  2. Polypeptide selon la revendication 1, dans lequel le résidu d'origine de la séquence de TGFβ1, TGFβ2 ou TGFβ3 humain natif est muté et remplacé par l'un des résidus d'acide aminé choisi dans le groupe comprenant :
    pour la position 30 : N, R, K, D, Q, L, S, P, V, I, G, C, T, A, E ;
    pour la position 32 : A ;
    pour la position 101 : A, E ;
    pour la position 51 : Q, W, Y, A ;
    pour la position 67 : H, F, Y, W et
    pour la position 6 : A.
  3. Polypeptide selon la revendication 2, dans lequel des mutations au niveau de l'interface d'interaction avec les récepteurs TGFβRII et/ou TGFβRIII sont ajoutées afin d'augmenter l'affinité du polypeptide pour les récepteurs.
  4. Polypeptide selon la revendication 2, couplé à un fragment d'IgG1 humaine comprenant les mutations ala234 et ala235 dans le domaine Cγ2 de l'IgG1 humaine.
  5. Polypeptide selon la revendication 1, dans lequel le polypeptide est choisi dans le groupe consistant en SEQ ID NO : 1, SEQ ID NO : 2, SEQ ID NO : 3, SEQ ID NO : 4, SEQ ID NO : 5, SEQ ID NO : 6, SEQ ID NO : 7, SEQ ID NO : 8, SEQ ID NO : 9, SEQ ID NO : 10, SEQ ID NO : 11, SEQ ID NO : 12, SEQ ID NO : 13, SEQ ID NO : 14, SEQ ID NO : 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO : 24, SEQ ID NO : 25, SEQ ID NO : 26, SEQ ID NO : 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQIDNO : 31, SEQ ID NO : 32, SEQ ID NO : 33, SEQ ID NO : 34, SEQ ID NO : 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO : 44, SEQ ID NO : 45, SEQ ID NO : 46, SEQ ID NO : 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO : 52, SEQ ID NO : 53, SEQ ID NO : 54, SEQ ID NO : 55, SEQ ID NO : 56, SEQ ID NO : 57, SEQ ID NO : 58, SEQ ID NO : 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO : 64, SEQ ID NO : 65, SEQ ID NO : 66, SEQ ID NO : 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO : 72 et SEQ ID NO : 73.
  6. Composition pharmaceutique appropriée pour le traitement du cancer, de la fibrose ou de maladies infectieuses chroniques, comprenant au moins un polypeptide selon la revendication 2 dans une plage entre 50ng/mL-10µg/mL, et un véhicule pharmaceutiquement approprié.
  7. Composition pharmaceutique selon la revendication 6, comprenant une quantité thérapeutiquement efficace d'un mélange des polypeptides de la revendication 2, et un véhicule pharmaceutiquement approprié.
  8. Composition pharmaceutique selon la revendication 6, dans laquelle le polypeptide est lié de manière covalente à une protéine porteuse.
  9. Composition pharmaceutique selon la revendication 8, dans laquelle la protéine porteuse est caractérisée en ce qu'elle est la région Fc d'immunoglobulines humaines.
  10. Composition pharmaceutique selon la revendication 8, dans laquelle le polypeptide est pégylé.
  11. Polypeptide selon l'une quelconque des revendications 1 à 5, destiné à être utilisé comme médicament.
  12. Polypeptide selon l'une quelconque des revendications 1 à 5, destiné à être utilisé dans le traitement du cancer, de la fibrose ou de maladies infectieuses chroniques.
  13. Polypeptide selon l'une quelconque des revendications 1 à 5, destiné à être utilisé dans le traitement du cancer ; dans lequel le polypeptide est destiné à améliorer la réponse cellulaire et/ou humorale à un vaccin thérapeutique contre le cancer.
EP13805228.7A 2012-11-09 2013-10-30 Polypeptides dérivés de tgf et leurs utilisations Active EP2918284B1 (fr)

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CU20120158A CU24181B1 (es) 2012-11-09 2012-11-09 POLIPÉPTIDOS DERIVADOS DEL TGFß
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PL238850B1 (pl) * 2017-10-19 2021-10-11 Calther Polska Spolka Z Ograniczona Odpowiedzialnoscia Aktywna forma transformującego czynnika wzrostu beta 1 (TGFβ1) do zastosowania w leczeniu pacjentów z glejakami wielopostaciowymi, w przypadku których to glejaków ponad 5% komórek nowotworowych jest EGFRvIII-pozytywna

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AU4572899A (en) * 1998-06-16 2000-01-05 Biogen, Inc. Variant type ii tgf-beta receptor fusion proteins and methods
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US6677432B1 (en) * 1998-10-07 2004-01-13 Stryker Corporation Mutations of the C-terminal portion of TGF-β superfamily proteins
ES2146552B1 (es) * 1998-11-24 2001-04-16 Inst Cientifico Tecnol Navarra Peptidos inhibidores de tgf/31
CA2575278A1 (fr) * 2004-06-30 2006-01-19 Molecular Logix, Inc. Antagonistes du recepteur du facteur de croissance epidermique et ses procedes d'utilisation
US7795389B2 (en) * 2004-09-28 2010-09-14 The Board Of Regents Of The University Of Texas System Antagonizing TGF-beta activity with various ectodomains TGF-beta receptors used in combination or as fusion proteins
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HK1213781A1 (zh) 2016-07-15
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ZA201504462B (en) 2018-11-28
KR102093494B1 (ko) 2020-03-26
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EA201590914A1 (ru) 2015-09-30
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US20150284441A1 (en) 2015-10-08
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CU24181B1 (es) 2016-04-25
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CA2887455C (fr) 2022-05-17
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